Spores are major mechanism by which the important nosocomial pathogen Clostridium difficile transmits disease, yet little is known about how this obligate anaerobe forms the resistant spore form. While preventing spore assembly would break the destructive cycle of infection and re-infection that characterizes C. difficile infections, no therapies currently target this developmental process. A major obstacle to developing such therapies is the absence of a basic understanding of how C. difficile forms a spore. Our long-term goal is to understand how C. difficile assembles the protective proteinaceous spore coat, which is necessary for functional spore formation. We recently identified the first spore morphogenetic proteins in the Clostridia as SpoIVA and SipL and showed that they directly interact. In this application, we aim to elucidate the molecular mechanisms by which SpoIVA and SipL coordinately regulate spore formation. We will use complementary genetic, biochemical, and cytological methods to determine the functional significance of SpoIVA-SipL complex formation during spore assembly, identify new SpoIVA and/or SipL interacting partners that regulate spore assembly, and localize SpoIVA and SipL during spore formation. By interrogating the mechanism by which SpoIVA and SipL interact and recruit coat proteins to the forespore during sporulation, the proposed studies will elucidate the functions of key regulators of spore formation, identify new morphogenetic proteins in the Clostridia, and develop new imaging methods for localizing proteins in live cells under anaerobic conditions. Since SipL is found exclusively in the Clostridia, these studies will expand our limited understanding of how spore assembly is regulated in the Clostridia. Completion of the proposed work will provide the mechanistic insight necessary for rationally designing screens that can identify C. difficile sporulation inhibitors and may also identify new potential therapeutic targets. As a result, these studies may lead to novel therapeutics that can prevent disease transmission by C. difficile and other clostridial spore-forming pathogens.